A realistic model of the structural organization of the major pathways of intermediary metabolism in the ciliate Tetrahymena pyriformis has been developed. This model will be subjected to further testing by adding leucine to the 9-substrate mixture used to develop the model. Use of (14C) leucine will allow introduction of CO2 compartmentation into the model and will provide new information on the organization of the pools of acetyl CoA. Paired flasks containing chlorpromazine (an inhibitory ligand of calmodulin) will also be studied, to assess the effects of inhibition of calmodulin-regulated systems in this early eukaryote. The data will include not only measurements of 14C flow into glycogen, CO2, and lipids, but also the concentrations and specific activities of selected intermediates such as glucose-6-phosphate and fructose-1, 6-diphosphate, thereby allowing not only quantitation of the complete flux pattern but also some insight into factors responsible for regulation of the pathways. The mathematical approach so far used, which involves extremely cumbersome algebraic manipulation will be improved by developing a matrix inversion procedure that will greatly facilitate the evaluation of alternate metabolic schemes. This procedure will be tested in conjunction with experiments designed to quantitate the flux of metabolites along the gluconeogenic/glycolytic/pentose phosphate pathways in hepatocytes isolated from fed rats and incubated with a mixture of glucose, mannose, ribose, glycerol, and acetate. These experiments will permit quantitation of the amount of futile cycling at key regulatory points of these pathways as well as establish the flux of metabolites through each step of the pentose phosphate pathway. The experiments will be performed in the presence of glucagon or of low concentrations of ethanol as well as presence of the 5-substrate mixture alone, thereby providing a quantitative picture of the effects of glucagon and of ethanol on hepatocyte metabolism. These experiments will also serve as the necessary first steps for development of models of hepatocyte metabolism that will include reactions occuring in the mitochondria and peroxisomes.